Almost 5 million women suffer from a urinary tract infection (UTI) each year. Urinary tract infection affects about 20% of women annually, and approximately 50% of women suffer from at least one bout of UTI in their lifetime, contributing significantly to hospital laboratory workloads. Following an ESCMID study carried out in 2001, it was shown that only 19% of hospitals within the EU carry out standard screening procedures as the norm. Asymptomatic bacteraemia in catheter patients are largely responsible for the increase in the number of antibiotic resistant organisms found in the hospital setting.
UTI is predominantly caused by a small range of organisms: Escherichia coli, Staphylococci, Proteus mirabilis, Enterococci, Kiebsiella; Bacillus (Gruenberg, 1980) and Candida albicans have also been implicated in UTI (Voss et al., 1994). Escherichia coli is the most common, however Candida infections are of particular concern as the organism is capable of ascending the urinary tract and it may lead to disseminated disease including renal disease. It is generally recognised that more than 105 bacteria/ml are needed to confirm the presence of urinary tract infection whereas values below 103 bacteria/ml are largely due to cross contamination. The development of a rapid method for detecting UTI, and identifying the causative organism is of primary importance, however, the availability of a generic method allowing improved detection of specific types of bacteria in body fluids would have wider medical significance. For example, the detection of bacteraemia (bacteria in blood) is of critical importance. There are additionally many non-clinical situations in which rapid differential diagnosis of microorganisms would be of significance, including detection of faecal indicator bacteria.
The role of a universal detection system for UTI organisms has already been demonstrated and a commercial kit based on nitrate detection is marketed in the USA. However this test is based upon a metabolic activity that is not present in all UTI organisms, for example Enterococcus spp. and therefore could give false negative results. Other limitations of this test are that false negative results may be produced through a lack of nitrates in a vegetarian diet or excess intake of ascorbic acid (Vitamin C). Urine also needs to be retained for 4-6 hours prior to the test. When trying to control a UTI infection the patient is advised to drink increased quantities of fluid thus posing a problem for urine retention.
When testing for the presence of a microorganism, it is common practice in microbiology to incorporate a hydrolytic chromogenic substrate into the growth medium, so that any organism expressing the appropriate enzyme for this substrate will hydrolyse it thereby releasing the coloured chromogen and acting as an indicator for the presence of a microorganism. This has commonly been carried out with X-D-galactosides and X-D-glucuronides, which detect coliforms and E. coli, respectively.
U.S. Pat. No. 5,221,606 discloses the use of chromogenic phenols, which have solubilising groups attached to them, such as carboxyl and quaternary ammonium groups, or both, which impart a certain degree of water solubility to their derived carboxylic ester derivatives.
Merely making a soluble chromogenic substrate is not enough however. It is important that the colour released on action of the enzyme remains localised with the bacterial colony that produced the enzyme, otherwise identification of bacteria would be difficult. Ideally a test would provide a chromogenic substrate which would impart a characteristic colour to different bacterial colonies.
We have demonstrated the selective absorption of chromophoric phenols (released from chromogenic substrates by microbial enzymatic activity) onto membranes in U.S. Pat. No. 5,221,606. It has been shown that several of the chromophores are tightly bound to types of membrane. Also, on binding, the intensity of the colour increases and may change (e.g. from red to blue), depending on the particular chromophore released.
By incorporating a rapid and cheap method of screening to replace the lengthy procedures currently being used, the accurate detection of a UTI and determination of the causative organism would enable appropriate antibiotic treatment to be given to patients at an early stage.
A common device for identifying substrates in body fluid is a dip-stick. There are two dip-stick formats used in a number of products on the market today. The most commonly used format is the lateral flow e.g. the pregnancy test. A typical lateral flow involves four main sections: sample application pad, conjugate release pad, analytical membrane and the wick (FIG. 1). Each section will have different characteristics such as absorption, wicking rates and binding properties thereby allowing a good flow through and clear results. The lateral flow format is generally used in the detection of compounds by antibodies and its main advantages are ease of use and speed.
The second format is the flow through which acts to concentrate the sample (FIG. 2). Greater volumes of fluid can be added to the test. The main disadvantage of the flow though is that in the majority of cases, especially those involving antibodies, a multi-step washing process needs to be carried out, thereby making it unsuitable for home testing.
The applicants have discovered that an improvement to the effectiveness of detection devices can be made by adding filtration means to the devices to separate differently sized microorganisms. Microorganisms can then be directed to discrete areas where they react with particular chromogens.